We propose to apply Raman difference spectroscopy to determine the nature of the structural changes at the hemes which are coupled to the quaternary conformational change associated with cooperative ligand binding to hemoglobin. We have shown with a very sensitive difference spectroscopic technique that several lines in both the high frequency range and low frequency range of the Raman spectrum are shifted in chemically and enzymatically modified hemoglobins in which the equilibrium between R and T states is altered. The high frequency lineshifts may be correlated with alterations in Pi electron density whereas the low frequency lineshifts may be correlated with changes in Fe-N ligand bonds and in position of porphyrin peripheral groups. We wish to determine whether the alterations in Pi electron density may be linked to these changes in heme structure and whether this linkage is dependent upon a quaternary conformational change or only upon a change in intrinsic affinity for ligands. To determine the relative contributions of the positions of peripheral groups of the hemes of the two different chains, we will reconstitute hemoglobins with meso- or deuteroheme introduced selectively into Alpha, Beta, or Gamma chains. By substituting Co(II)- for Fe(II)-prophyrin, we may investigate the contributions of the metal-ligand bonds in the two chains to the Raman spectra. To determine the effects of quatenary structural change on the spectra of liganded hemoglobins, we will study carp oxy- and CO-hemoglobin, which can be switched from R to T by inositol hexaphosphate or low pH. We hope to sort out contributions of alterations in the 4 degree conformational equilibrium constant, L, from changes in intrinsic ligand affinity of the states, KT and KR, by studying hemoglobins in which these values are different from those in human hemoglobin A. We will examine the spectra of cat hemoglobin B, in which the value of L is especially large, human fetal hemoglobin FI and FII, in which the T state is also unusually stable, and hemoglobins in which the inorganic anion binding sites in the deoxytetramer are blocked with o-iodobenzoic acid or glutarylsalicylamide.